Japan has become an aging society, with the average life span in Japan being the longest in the world. People now focus on the quality of life (QOL), that is, living a better life, rather than simply living longer. Speaking and eating are important functions especially for elderly people, which lead to a reason for living. In that sense, health maintenance for masticatory organs, including maintenance of teeth, can be said to be a factor that significantly influences QOL. Mastication is a function indispensable for ingestion of food. Moreover, it is becoming clear, from recent studies on the mastication system, that mastication influences various systemic functions in the entire body, for example, mastication stimulates brain cells to promote mental/neural development and activation, enhances the immune function, and furthermore, suppresses obesity. Thus, a decrease in the masticatory function due to loss of teeth may lead to occurrence of dementia, lifestyle-related diseases, and the like.
Teeth are exposed to the oral cavity breaking through the epithelium, and continuity of the epithelium disappear at the boundaries between teeth and gingiva. Thus, teeth are in a very special environment in the body. Teeth and gingiva are constituted by “epithelial attachment” and “connective tissue attachment”. In the former attachment, an epithelium called junctional epithelium adheres to the tooth surface (enamel) through hemidesmosomes and the basal lamina. The latter attachment is constituted by the periodontal ligament. Collagen fibers are inserted into root surface cementum while their calcification proceeds, and those fibers are inserted into alveolar bone similarly while their calcification proceeds, providing gingival fibers. By this, teeth are strongly bound to alveolar bone and gingiva.
The periodontal ligament is an aggregate of periodontal ligament fibers, and, as described above, those fibers run almost perpendicularly to the tooth surface and the alveolar bone surface in most cases. The run of those fibers plays a role as a cushion that reduces the force applied to the teeth. It reduces mechanical loads on the teeth such as the force due to improper occlusion, or bruxism. The periodontal ligament also plays a role in transmitting a delicate sense felt upon chewing to the brain. By this, hardness of the object chewed can be recognized, and hence an object can be chewed with an appropriate force, so that unnecessary injury of teeth and gingiva can be avoided. The periodontal ligament is rich in blood vessels, and has abundant supply of blood. Therefore, during inflammation, the periodontal ligament can protect against infection by recruiting leukocytes.
Methods using a denture or a bridge have been mainly used for replacing a tooth that was lost by dental caries, periodontal disease, or the like. However, in recent years, therapeutic methods using a dental implant, which is aesthetic and functional, and does not require cutting preparation of adjacent teeth, are attracting attention. The implant treatment means a treatment in which a titanium or titanium alloy support is embedded in the jawbone in a place where a tooth is lost, and the artificial tooth is repaired using the support as a base. According to a recent survey, implant treatment has become a common dental treatment in Japan (Non-patent Document 1), received by 1 or 2 out of 50 adults who are 30 years of age or older. An implant is composed of a fixture (artificial tooth root), an abutment (connecting portion), and an upper structure (artificial tooth crown). In the basic flow of implant treatment, the fixture section of the implant is embedded in the bone, and, after sufficient adhesion of the fixture to the bone, a crown is formed on the upper structure. The process of adhesion of the fixture to the bone is called osseointegration, and it usually requires a period of 2 to 6 months. In cases where an operation for increasing bone or the like is necessary before embedding the fixture, a period of not less than one year is sometimes required. The ten-year implant survival rate is almost 90% or more, which is influenced by the age of the patient and implant system selection, and the like. The patients who visit implant treatment facilities are young on an average, but some data also show that patients who receive the treatment most frequently are in the age groups of forties and fifties that have significant influence of periodontal disease and the like.
However, various problems have also been pointed out for the implant method. For successful implant treatment, it is important to achieve sufficient adhesion of the implant to the bone, and to allow stabilization of the implant in the jawbone so that movement of the implant does not occur therein. There are a significant number of cases where implantation failed due to failure in achieving such stabilization. In a physiologically normal state, a connective tissue called periodontal ligament tissue is present in the vicinity of a tooth. Since, as described above, the periodontal ligament tissue fills the space between the tooth and the alveolar bone to function as a cushion material, the mechanical load on the tooth can be reduced, and inflammation that occurred in the vicinity of the tooth can be ameliorated, so that influences on the surrounding bone can be suppressed.
In current implant treatment, a biocompatible metal such as titanium is screwed into the jawbone, so that the implant is fixed in a state where the metal is in contact with, and adhering to, the bone. In this method, there is no periodontal ligament tissue surrounding the implant, and angiogenesis is unlikely to occur. Therefore, once inflammation occurs, migration of immune cells to suppress the inflammation is difficult, and the inflammation is likely to show direct and extensive spread to the bone (peri-implantitis) (Non-patent Document 2). Moreover, an implant is likely to cause detachment of gingiva because of the absence of the connective tissue attachment, and inflammation easily progresses once it occurs, leading to difficulty in curing of the inflammation. There is no appropriate therapeutic method for such symptoms, which is problematic.
When the development of the implant initially started, the fixture section had a smooth surface, and it was called the cylinder type. Thereafter, it was found that a screw-shaped fixture section is advantageous in the initial fixation, so that the current implant is of a type having a screw thread. More recently, an implant having a fixture section coated with HA (hydroxyapatite) was developed in order to allow faster and stronger binding to bone. Since HA has components similar to components in the living body, it can be expected to show bone-conducting capacity in osteogenesis. There is actually a result of an experiment which shows that, while an HA-coated implant shows formation of bone to a distance of 1.5 mm around the implant, an HA-uncoated implant (with a titanium surface) allows formation of bone to a distance of up to only 0.3 mm around the implant. In Japan, a variety of production methods have been developed in 1990s. In particular, implants coated with recrystallized HA have been widely applied to clinical use as implants with which early bone induction can be expected. More recently, it was found that a stronger bond to the bone can achieved by surface treatment with a strong acid rather than by blasting. After this discovery, surface properties of implants have been improved by preparation of rough surfaces (micro-rough structures) on implants by blasting or strong acid treatment. Currently, attempts are being made to cause osteoconduction and calcification by fluorine coating of the implant surface to thereby accelerate the cure, but no implant has completely solved the problems so far.
On the other hand, studies on treatment of periodontal disease, which is considered to be a cause of tooth loss, by transplantation of cells are being actively carried out in recent years. In many of these studies, single cells are plated on a three-dimensional matrix, and the matrix is then implanted by injection into a site where a tissue is lost. For example, Non-patent Document 3 describes that, by culturing cells separated from periodontal ligament in the vicinity of a titanium implant to allow formation of a plurality of cell layers on the implant surface, and then inserting the implant in an affected site, alveolar bone and the like could be formed in the vicinity of the implant. However, construction of an expected tissue has not been realized yet. Possible reasons for this failure are difficulties in selection of the cell source, and in control of localization of cell differentiation in the site where a tissue is lost. For regeneration of a periodontal tissue, cementum needs to be newly generated on the root surface. Therefore, not only the periodontal ligament, which is a soft tissue, but also the cementum or the alveolar bone, which is a hard tissue, need to be regenerated at the same time to allow their functional connection to each other. If these tissues are formed at different times depending on actions of progenitor cells and growth factors specific to the tissues, the cell transplantation method in the regeneration of the periodontal tissue needs to be more delicate. That is, the transplantation requires definition of places where cells are to be arranged, and arrangement of appropriate cells in each of those places, rather than simply injecting single cells to the lost portion after space making, and allowing tissue differentiation in the living body.
Preparation of the cells required in this process has been carried out by culture on a glass surface, or on a surface of a synthetic polymer subjected to various treatments. For example, various containers and the like made of polystyrene subjected to surface treatment such as γ-ray irradiation or silicone coating are commonly used as cell culture vessels. Cells cultured/grown using such cell culture vessels are detached and collected from the surface of the vessels by treatment with a protease such as trypsin, or a chemical agent. However, collection of grown cells by the chemical treatment described above has been pointed out to have drawbacks such as a laborious treatment process, high possibility of contamination, and possible degeneration or damaging of the grown cells by the chemical treatment, which may lead to deterioration of intrinsic functions of the cells.
In order to overcome such drawbacks, several techniques have been proposed so far. In particular, Patent Document 1 enabled preparation of a cell sheet having sufficient strength by culturing anterior ocular segment-related cells on a cell culture support formed by coating the surface of a base material with a temperature responsive polymer of which upper or lower critical solution temperature against water is 0 to 80° C., layering, if necessary, a cultured cell layer thereon by a conventional method, and then detaching the cultured cell sheet simply by changing the temperature of the support. The cell sheet also retains basal lamina-like protein, and shows obviously better engraftment on a tissue compared to the dispase-treated cells described above. In Patent Document 2, it was discovered that a cardiac muscle-like cell sheet can be constructed by culturing cardiac muscle tissue cells on a cell culture support of which surface is grafted with a temperature responsive polymer. It was also discovered that the cell sheets have less structural defects and several functions as a cardiac muscle-like tissue in vitro by changing the temperature of the culture medium not to less than the upper critical solution temperature or not more than the lower critical solution temperature, bringing the layered cell sheet into close contact with a polymer membrane, detaching the cell sheet together with the polymer membrane, and then forming the resultant three-dimensional structure by a predetermined method.
Patent Document 3 shows that, when periodontal ligament tissue cells are cultured on a cell culture support of which surface is grafted with a temperature responsive polymer to obtain a periodontal ligament cell sheet, the cultured periodontal ligament cell sheet can be detached by changing the temperature of the culture medium not to less than the upper critical solution temperature or not more than the lower critical solution temperature. And it also shows that regeneration of a periodontal tissue containing a periodontal ligament tissue can be induced by attaching the periodontal ligament cell sheet to the root of a natural tooth. Patent Document 4 describes a method for regenerating a periodontal tissue by wrapping the crown or the root of a natural tooth with a periodontal ligament cell sheet. However, none of these methods have been carried out aiming to solve the problems on engraftment and fixation of implants made of a metal such as titanium.